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The extracellular domain (EC) of pemphigus vulgaris antigen (PVA) mediates weak homophilic adhesion
Oct. 31IMiniSymposimn
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227
1 - Adhesion Molecules in Skin Structure and Inflammation
230 DISTRIBUTION AND CHARACTERJZATION OF ECADHERIN EXPRESSED BY MURINE DENDRITIC CELLS. p VW .I MC U&y, Dermatology Branch, NCI, Bethesda, MD. USA Recent studies indicate that murine Langwham cells (LC) synthesize and express E-cadherin (E-cad) that mediates adhesion of LC to normal keratincytes in vitro. To determine if Ecad is selectively expressed by LC as compared with other dendritic cells (DC). we isolated DC from various tissues and assessed expression of E-cad and DC surface antigens by flow cytometry. DC from BALBlc spleen and thymus were released with collagenase, floated on 35% BSA gradients and additionally enriched na transient adherence to plastic. DC from inguinal and axillxy (skin-associated) or mesenteric (gut-associated) lymph nodes (LN) were released with collagenase. and floated on 14.5% metrizamide gradients. Cells were analyzed after staining with antiE-cad mAb (or mAb reactive with DC) and appropriate secondary Ab. Preparations enriched in splenic DC and DC from gut-associated LN were devoid of E-cad expressing cells. Some DC from thymus and skin-associated LN appeared to express low levels of E-cad, however. We also cultured blood DC from mace treated with cyclophosphamide (200 mgikg IP) 8 days before phlebotomy. After 10 days in media supplemented wth GM-CSF (10 “g/ml), we recovered DC functtonally and phenotypically identical to those described by Inaba and coworkers (J. Exp. Med. 175:1157,1992). Blood DC expressed immunoreactive Ecad in addition to DC surface antigens. We isolated blood DC RNA and detected mRNA encoding portions of both extracellular and intracellular regions of E-cad using E-cad specific primers and RTPCR. E-cad immunoprecipitated from blood DC also comigrated with that from fibroblasts transfected with E-cad cDNA. These resulu indicate that among DC, E-cad is selectively expressed by LC. cells that may give rise to LC (blood DC), cells that may be derived from LC (skin-associated LN DC) and cells that may be closely related to LC (thymic DC). These studies also confirm that E-cad expressed by murine DC is identical to that expressed by other cells.
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CADHERIN DEPENDENT REGULATORY MECHANISMS OF VASCULAR ENDOTHELIAL CELL-CELL INTERACTIONS. &&& * lmamura, Department of Dermatology. Kyoto University Faculty of Medicine, Kyoto, Japan Cadherins are calcium dependent cell adhesion molecules, playing a key role in cell-cell mteractiona of various kinds of cells. However the properttes of vascular endothelial cell (VEC) cadherin are not well known. To learn more about the molecules, we established the monoclonsl antibody (ENCDl) which specifically inhibit the cell-cell adhesion of VEC. by immuntzation of murine endothelial cell line (FZ) treated with trypsin containing 1mM Ca++(TC). ENCDl had followmg characters. (I) It caused the cell-cell detachment of endothelnl cells. (2) lmmunocytochemistry demonstrated that cell-cell borders of F2 cells were specifically stained, whereas those of other cell tytxs were not (3) Immunoblot analvsis showed the single band wth a mol&x weight of 13OKd. This pro&n band was pr&rved by TC treatment. but not bv TE ttrvosin wtth ImM EGTA) treatment. These data clexly demonstrated that the antigen wab VEC specific cadherin. To clarify It\ f&ion. the cell-cell ndhc\iv&e,\ and the fell barrier propertxs w&e measured. A co-cultivation of ENCDI with F2 cells Induced the cellular dn>ocintlon m cell aggregation awy and dcrtroyed the honeycomb stntcture on Matrl-gel. Furthermore ENCDI dlsrupted the barrier propaws in DAB twmeabllitv ;ISGIY. There observations indicate that VEC soeclfic cadherin plays a c&al rolk in cellulx integrity and permeability.
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THE EXTRACELLULAR DOMAIN (EC, OF PEMPHlCUS “ULGARlS ANTlGEN (PVAI MEDIATES WEAK HOMOPHILIC ADHESION. Maaavuki~. Sarolta. Kar~atl. Vera Klaus-Kovtun. Mark C. “de”. lohn R. Stanley. Dermatology Branch, NIH, Bethesda. MD. USA PVA IS in the aupergene family of cadherins. transmembrane protems that mcdmte calcium-dependent homoph~llc ceil adhesion. lmtially described cadherim, such as E-cadher,” (&ad,, mediaw adhesmn when transfected lnto mouse flbroblaats (L cells) only of ,he,r mtracellular region (10 interacls wth the cytoskeleton through bmding to a. p. and y catcnms. which are presenl in L cells. This rytoskeletal interactmn IS thought 10 cluster the moleculea m the membrane, thereby increasing cadhenn awdity. To determme of the PVA EC mediates adhesmn and to compare II to the Ecad EC. we transfected L cells wth Ecad cDNA or with a chlmerw cDNA encoding the PVA EC and the Ecad IC (PVC&,, Both encoded proteins were expressed on the cell surface, as assessed by flow cytometry wth anti~Ecad EC mono~lonal antGxdy and PV sera. The IC of both molecules bound the carenins equally as shown by their co-m~munoPrecip~tauon wth Ecad and PVCad. When smgle cell suspensions. &tamed by EDTA. were allowed to aggregate I” I mM calciw,,. the Ecad cell, ahawed strong aggregaho” wnh large clumPa. whereas the PVCad cells formed Small aggregates that could be easily dwupted However. the PVCad aggregation was speafx and homophlhc because when PVCad cells were mlred with conlrol L cellv transfected with the neomycmresistance gene. and one Population was labeled wth a fluorescent cytoplasmic dye, only PVCad cells were m the aggregates. We conclude that the PV EC functions weakly m homophllx adheamn, and speculate that the cytoplasmx factors promoung adheslo” may be dlflerent for Ecad and PVA.
50
227
1 - Adhesion Molecules in Skin Structure and Inflammation
230 DISTRIBUTION AND CHARACTERJZATION OF ECADHERIN EXPRESSED BY MURINE DENDRITIC CELLS. p VW .I MC U&y, Dermatology Branch, NCI, Bethesda, MD. USA Recent studies indicate that murine Langwham cells (LC) synthesize and express E-cadherin (E-cad) that mediates adhesion of LC to normal keratincytes in vitro. To determine if Ecad is selectively expressed by LC as compared with other dendritic cells (DC). we isolated DC from various tissues and assessed expression of E-cad and DC surface antigens by flow cytometry. DC from BALBlc spleen and thymus were released with collagenase, floated on 35% BSA gradients and additionally enriched na transient adherence to plastic. DC from inguinal and axillxy (skin-associated) or mesenteric (gut-associated) lymph nodes (LN) were released with collagenase. and floated on 14.5% metrizamide gradients. Cells were analyzed after staining with antiE-cad mAb (or mAb reactive with DC) and appropriate secondary Ab. Preparations enriched in splenic DC and DC from gut-associated LN were devoid of E-cad expressing cells. Some DC from thymus and skin-associated LN appeared to express low levels of E-cad, however. We also cultured blood DC from mace treated with cyclophosphamide (200 mgikg IP) 8 days before phlebotomy. After 10 days in media supplemented wth GM-CSF (10 “g/ml), we recovered DC functtonally and phenotypically identical to those described by Inaba and coworkers (J. Exp. Med. 175:1157,1992). Blood DC expressed immunoreactive Ecad in addition to DC surface antigens. We isolated blood DC RNA and detected mRNA encoding portions of both extracellular and intracellular regions of E-cad using E-cad specific primers and RTPCR. E-cad immunoprecipitated from blood DC also comigrated with that from fibroblasts transfected with E-cad cDNA. These resulu indicate that among DC, E-cad is selectively expressed by LC. cells that may give rise to LC (blood DC), cells that may be derived from LC (skin-associated LN DC) and cells that may be closely related to LC (thymic DC). These studies also confirm that E-cad expressed by murine DC is identical to that expressed by other cells.
228
231
CADHERIN DEPENDENT REGULATORY MECHANISMS OF VASCULAR ENDOTHELIAL CELL-CELL INTERACTIONS. &&& * lmamura, Department of Dermatology. Kyoto University Faculty of Medicine, Kyoto, Japan Cadherins are calcium dependent cell adhesion molecules, playing a key role in cell-cell mteractiona of various kinds of cells. However the properttes of vascular endothelial cell (VEC) cadherin are not well known. To learn more about the molecules, we established the monoclonsl antibody (ENCDl) which specifically inhibit the cell-cell adhesion of VEC. by immuntzation of murine endothelial cell line (FZ) treated with trypsin containing 1mM Ca++(TC). ENCDl had followmg characters. (I) It caused the cell-cell detachment of endothelnl cells. (2) lmmunocytochemistry demonstrated that cell-cell borders of F2 cells were specifically stained, whereas those of other cell tytxs were not (3) Immunoblot analvsis showed the single band wth a mol&x weight of 13OKd. This pro&n band was pr&rved by TC treatment. but not bv TE ttrvosin wtth ImM EGTA) treatment. These data clexly demonstrated that the antigen wab VEC specific cadherin. To clarify It\ f&ion. the cell-cell ndhc\iv&e,\ and the fell barrier propertxs w&e measured. A co-cultivation of ENCDI with F2 cells Induced the cellular dn>ocintlon m cell aggregation awy and dcrtroyed the honeycomb stntcture on Matrl-gel. Furthermore ENCDI dlsrupted the barrier propaws in DAB twmeabllitv ;ISGIY. There observations indicate that VEC soeclfic cadherin plays a c&al rolk in cellulx integrity and permeability.
,,
1
229
THE EXTRACELLULAR DOMAIN (EC, OF PEMPHlCUS “ULGARlS ANTlGEN (PVAI MEDIATES WEAK HOMOPHILIC ADHESION. Maaavuki~. Sarolta. Kar~atl. Vera Klaus-Kovtun. Mark C. “de”. lohn R. Stanley. Dermatology Branch, NIH, Bethesda. MD. USA PVA IS in the aupergene family of cadherins. transmembrane protems that mcdmte calcium-dependent homoph~llc ceil adhesion. lmtially described cadherim, such as E-cadher,” (&ad,, mediaw adhesmn when transfected lnto mouse flbroblaats (L cells) only of ,he,r mtracellular region (10 interacls wth the cytoskeleton through bmding to a. p. and y catcnms. which are presenl in L cells. This rytoskeletal interactmn IS thought 10 cluster the moleculea m the membrane, thereby increasing cadhenn awdity. To determme of the PVA EC mediates adhesmn and to compare II to the Ecad EC. we transfected L cells wth Ecad cDNA or with a chlmerw cDNA encoding the PVA EC and the Ecad IC (PVC&,, Both encoded proteins were expressed on the cell surface, as assessed by flow cytometry wth anti~Ecad EC mono~lonal antGxdy and PV sera. The IC of both molecules bound the carenins equally as shown by their co-m~munoPrecip~tauon wth Ecad and PVCad. When smgle cell suspensions. &tamed by EDTA. were allowed to aggregate I” I mM calciw,,. the Ecad cell, ahawed strong aggregaho” wnh large clumPa. whereas the PVCad cells formed Small aggregates that could be easily dwupted However. the PVCad aggregation was speafx and homophlhc because when PVCad cells were mlred with conlrol L cellv transfected with the neomycmresistance gene. and one Population was labeled wth a fluorescent cytoplasmic dye, only PVCad cells were m the aggregates. We conclude that the PV EC functions weakly m homophllx adheamn, and speculate that the cytoplasmx factors promoung adheslo” may be dlflerent for Ecad and PVA.